WO2022204321A1 - Évaluation concurrente conservatrice de modifications de l'adn - Google Patents

Évaluation concurrente conservatrice de modifications de l'adn Download PDF

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WO2022204321A1
WO2022204321A1 PCT/US2022/021602 US2022021602W WO2022204321A1 WO 2022204321 A1 WO2022204321 A1 WO 2022204321A1 US 2022021602 W US2022021602 W US 2022021602W WO 2022204321 A1 WO2022204321 A1 WO 2022204321A1
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dna
dna fragment
nucleic acid
acid sample
sample
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Brady CULVER
Daniella BIANCHI-FRIAS
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Ambry Genetics Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/10—Characterised by chemical treatment
    • C12Q2523/125—Bisulfite(s)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the methods relate to determining whether a DNA has been modified by evaluating DNA modification status with respect to a single sample.
  • epigenetics is the study of heritable phenotype changes that do not involve changes in DNA sequences.
  • the epigenetic changes involve changes that affect gene activity and expression.
  • Epigenetics also refers to the changes that may be functionally relevant to the genome and involve chemical modifications of nucleic acids. Examples of the chemical modifications include methylation, hydroxy methylation, and strand specific DNA deamination. These chemical modifications do not change the gene sequence, but are known to affect gene expression. These epigenetic changes may last through for the duration of the cell’s life, or even through the cell lineage.
  • cfDNA cell-free DNA
  • cfDNA The disadvantage of cfDNA is the relatively low amount of cfDNA present within the blood sample of a patient. For instance, a typical cfDNA concentration is as low as 1 ng/ml. Alborelli I. et al. (2019). This relatively low amount impairs the ability of researchers and clinicians to collect enough material for testing or detecting the presence of DNA modifications. Therefore, the ability to conduct multiple analyses on the same sample is extremely useful.
  • the present disclosure provides methods for performing a medical procedure by determining the mutation and DNA modification status of a nucleic acid sample from a subject with respect to a single sample.
  • the present disclosure also provides methods for diagnosis by determining whether a DNA of a nucleic acid sample contains nucleobase modifications and mutations, by assessing the DNA modification and mutation status with respect to a single sample.
  • This disclosure sets forth processes, in addition to making and using the same, and other solutions to problems in the relevant field.
  • a method for assessing DNA modification status of a nucleic acid sample from a subject with respect to a single sample comprising: (a) a step of evaluating DNA modification status of a DNA fragment in a nucleic acid sample in order to assess the DNA modification status of a biological samples.
  • the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample before, during, or after step (a).
  • the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample before step (a).
  • the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample during step (a). In certain embodiments, the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample after step (a). In certain embodiments, the method comprises (c) a step of producing a copied DNA fragment of the DNA fragment in the nucleic acid sample before step (a). In certain embodiments, the method comprises in step (a) the DNA modification status of the DNA fragment in the nucleic acid sample is evaluated basing on methylations, hydroxy methylations, strand specific deaminations or presence of N- methyladenine bases occurred to the DNA fragment.
  • the method in step (b), DNA mutation status of the DNA fragment in the nucleic acid sample is evaluated basing on single nucleotide variations, insertions, translocations, copy number, or deletions present in the DNA fragment.
  • the method in step (c), the copied DNA fragment is produced by immobilizing the DNA fragments to a substrate by: (a) immobilizing the DNA fragment to a substrate to create an immobilized DNA fragment; (b) copying the DNA fragment through PCR amplification to create a copied DNA fragment; and (c) separating the copied DNA fragment from the immobilized DNA fragment.
  • the method comprises each strand of the DNA fragment is immobilized separately to the substrate.
  • the method comprises a step (d) of obtaining a nucleic acid sample from the subject before step (a). In certain embodiments, the method comprises a step (e) of tagging a DNA fragment in the nucleic acid sample to create a tagged DNA fragment. In certain embodiments, step (e) is performed before the DNA fragment is copied, immobilized to a substrate, or before evaluating DNA mutation status. In certain embodiments, the method comprises a step of evaluating DNA mutation status of the copied DNA fragment.
  • a method for performing a medical procedure by assessing DNA modification status of a nucleic acid sample from a subject with respect to a single sample comprising: (a) a step of evaluating DNA modification status of a DNA fragment in a nucleic acid sample in order to assess the DNA modification status of a biological samples.
  • the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample before, during, or after step (a).
  • the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample before step (a). In certain embodiments, the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample during step (a). In certain embodiments, the method comprises (b) a step of evaluating DNA mutation status of the DNA fragment in the nucleic acid sample after step (a).
  • the method comprises (c) a step of producing a copied DNA fragment of the DNA fragment in the nucleic acid sample before step (a).
  • the method comprises in step (a) the DNA modification status of the DNA fragment in the nucleic acid sample is evaluated basing on methylations, hydroxy methylations, strand specific deaminations or presence of N-methyladenine bases occurred to the DNA fragment.
  • the method in step (b) DNA mutation status of the DNA fragment in the nucleic acid sample is evaluated basing on single nucleotide variations, insertions, translocations, copy number, or deletions present in the DNA fragment.
  • the method in step (c), the copied DNA fragment is produced by immobilizing the DNA fragments to a substrate by: (a) immobilizing the DNA fragment to a substrate to create an immobilized DNA fragment; (b) copying the DNA fragment through PCR amplification to create a copied DNA fragment; and (c) separating the copied DNA fragment from the immobilized DNA fragment.
  • the method comprises each strand of the DNA fragment is immobilized separately to the substrate.
  • the method comprises a step (d) of obtaining a nucleic acid sample from the subject before step (a).
  • the method comprises a step (e) of tagging a DNA fragment in the nucleic acid sample to create a tagged DNA fragment.
  • step (e) is performed before the DNA fragment is copied, immobilized to a substrate, or before evaluating DNA mutation status.
  • the method comprises a step of evaluating DNA mutation status of the copied DNA fragment.
  • a method for assessing DNA modification status of a biological sample from a subject with respect to a single sample from a subject suspected of having a disease comprising: obtaining a nucleic acid sample from the subject.
  • the method comprises tagging a DNA fragment of the nucleic acid sample from the subject to create a tagged DNA fragment.
  • the method comprises immobilizing the tagged DNA fragment to a substrate, wherein each strand of the tagged DNA fragment in the nucleic acid sample is bound separately to the substrate.
  • the method comprises evaluating DNA mutation status of the tagged DNA fragment.
  • the method comprises evaluating DNA modification status of the tagged DNA fragment.
  • the method comprises assessing the DNA modification status of the nucleic acid sample.
  • a method for performing a medical procedure by assessing DNA modification status of a nucleic acid sample from a subject with respect to a single sample from a subject suspected of having a disease comprising: obtaining a nucleic acid sample from the subject.
  • the method comprises tagging a DNA fragment of the nucleic acid sample from the subject to create a tagged DNA fragment.
  • the method comprises immobilizing the tagged DNA fragment to a substrate, wherein each strand of the tagged DNA fragment in the nucleic acid sample is bound separately to the substrate.
  • the method comprises evaluating DNA mutation status of the tagged DNA fragment.
  • the method comprises evaluating DNA modification status of the tagged DNA fragment.
  • the method comprises assessing the DNA modification status of the nucleic acid sample.
  • a method for diagnosing a patient with a potential disease by evaluating DNA modification status of a nucleic acid sample from a subject with respect to a single sample from a subject suspected of having a disease comprising: obtaining a nucleic acid sample from a subject.
  • the method comprises tagging a DNA fragment of the nucleic acid sample from the subject to create a tagged DNA fragment.
  • the method comprises immobilizing the tagged DNA fragment to a substrate, wherein each strand of the tagged DNA fragment in the nucleic acid sample is bound separately to the substrate.
  • the method comprises evaluating DNA mutation status of the tagged DNA fragment.
  • the method comprises evaluating DNA modification status of the tagged DNA fragment.
  • the method comprises diagnosing a subject with a potential disease by evaluating the DNA modification status of the nucleic acid sample.
  • a method for performing a medical procedure by diagnosing a patient with a potential disease by evaluating DNA modification status of a nucleic acid sample from a subject with respect to a single sample from a subject suspected of having a disease, the method comprising: obtaining a nucleic acid sample from a subject.
  • the method comprises tagging a DNA fragment of the nucleic acid sample from the subject to create a tagged DNA fragment.
  • the method comprises immobilizing the tagged DNA fragment to a substrate, wherein each strand of the tagged DNA fragment in the nucleic acid sample is bound separately to the substrate.
  • the method comprises evaluating DNA mutation status of the tagged DNA fragment.
  • the method comprises evaluating DNA modification status of the tagged DNA fragment. In certain embodiments, the method comprises diagnosing a subject with a potential disease by evaluating the DNA modification status of the nucleic acid sample. [0014] In some embodiments, in any of the previous embodiments, the method further comprises copying the tagged cfDNA molecule through PCR amplification to produce an untagged copy; binding the tagged cfDNA molecule to the substrate; separating the tagged cfDNA from the untagged copy; and analyzing the mutation status of the unbound material.
  • FIG. 1 Exemplary method of analyzing cfDNA.
  • a pool of nucleic acids is extracted from a biological source to obtain the cfDNA.
  • a methylated cytosine residue on the cfDNA is depicted.
  • a first mutation, SNV or indel, present on both strands of a duplex fragment of cfDNA is depicted.
  • a second mutation, SNV or indel, present on both strands of a duplex fragment of cfDNA is depicted.
  • an adapter which contains an anchoring sequence that is to be ligated to the cfDNA is depicted.
  • the adapter can have various functional elements comprised within its sequence: methylated C residues, UMI, or primer binding sites.
  • a Y- shaped adapter is depicted.
  • an affinity tag present on one of the strands of the adapter is depicted.
  • the affinity tag is at the 5' end of the adapter and is a biotinylated nucleotide.
  • cfDNA is ligated to the adapter depicted in 5.
  • a pool of adapter-cfDNA ligation constructs resulting from the ligation reaction in 7 is depicted.
  • a bead or other solid phase matrix with ability to bind to the affinity tag in 6 which in turn is bound to cfDNA molecules is depicted. Fragments from elements 2, 3, and 4 are bound to the bead and are in their native unmodified state.
  • the polymerase, free dNTP, ions, and buffer are added to the immobilized cfDNA constructs to perform a primer extension reaction on the bead.
  • a primer and first extension product are depicted. The primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents extension from the primer towards to bead.
  • a primer and second extension product are depicted.
  • the primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents extension from the primer towards to bead.
  • a primer and third extension product are depicted.
  • the primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents extension from the primer towards to bead.
  • primer extension products from on-bead primer extension reaction is depicted. These products are not bound by the affinity matrix. These products faithfully pass on the identity of elements 3 and 4, but not 2.
  • primer extension products the copies of the biological DNA template, are detected or read by various methodologies.
  • This example focuses on DNA sequencing instruments.
  • bead bound DNA, 9, is treated with an agent that alters its sequence, in this example, sodium bisulfite treatment is performed to deaminate unmethylated cytosines.
  • DNA altering agent e.g. sodium bisulfite is depicted.
  • a first product of DNA altering treatment which retains the identity of the methylated cytosine is depicted.
  • a second product of DNA altering treatment which converts unmethylated cytosine to uracil is depicted.
  • a third product of DNA altering treatment which converts unmethylated cytosine to uracil is depicted.
  • a primer and fourth extension product are depicted.
  • the primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents extension from the primer towards the bead.
  • the extension product incorporates the complement of the methylated C residue, G, as it was not converted to uracil.
  • a primer and fifth extension product is depicted.
  • the primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents an extension from the primer towards to bead.
  • the extension product incorporates the complement of the Uracil (produced by conversion of the unmethylated C residue), A.
  • a primer and sixth extension product are depicted.
  • the primer is complementary to and hybridizes with an element within the free 3’ end of the ligated adapter.
  • the dotted arrow represents an extension from the primer towards to bead.
  • the extension product incorporates the complement of the Uracil (produced by conversion of the unmethylated C residue), A.
  • a pool of extension products, 21, 22, and 23 are depicted.
  • extension products from altered DNA can be detected or read by various methodologies. This depiction focuses on DNA sequencing instruments, but can be applied to any other detection method.
  • FIG. 2 Alternative method of analyzing cfDNA.
  • the products of amplification of 8 (FIG. 1) are depicted.
  • the template molecules retain original adapter, 5, and affinity tag, 6, while amplification products do not.
  • the amplification product, 26, is mixed with affinity matrix to separate original tagged biological DNA, 8, from amplification products is depicted.
  • unbound constructs comprising the amplification products of 8 are depicted. These constructs retain information about DNA sequence identity, but not modification status, e.g. methylation.
  • FIG. 3 Barplot showing measured methylation percent of a BRCA1 promoter locus in OVCAR8 or SW620 DNA following enzymatic conversion of unmethylated Cytosine to Uracil.
  • the “ enz beads” suffix refers to material that had been labeled with biotinylated adapter at ligation, amplified by PCR, and then captured on Streptavidin beads for the cytosine conversion step.
  • the “ enz ctrl” suffix refers to material that was not amplified or captured on beads.
  • the low %Methylation observed for SW620 material is an indicator of the efficiency of Cytosine conversion as this material is expected to lack any methylation at BRCA1 promoter. The expectation was to see 66% methylation for OVCAR8 cell line, which is represented by an approximately 1 Ct difference between methylated and unmethylated product.
  • biological sample refers to a sample derived from, obtained by, generated from, provided from, take from, or removed from an organism; or from fluid or tissue from the organism.
  • Biological samples include, but are not limited to synovial fluid, whole blood, blood serum, blood plasma, urine, sputum, tissue, saliva, tears, spinal fluid, tissue section(s) obtained by biopsy, cell(s) that are placed in or adapted to tissue culture, sweat, mucous, fecal material, gastric fluid, abdominal fluid, amniotic fluid, cyst fluid, peritoneal fluid, pancreatic juice, breast milk, lung lavage, marrow, gastric acid, bile, semen, pus, aqueous humor, transudate, and the like including derivatives, portions and combinations of the foregoing.
  • biological samples include, but are not limited, to blood and/or plasma.
  • biological samples include, but are not limited, to urine or stool.
  • Biological samples include, but are not limited, to saliva.
  • Biological samples include, but are not limited, to tissue dissections and tissue biopsies.
  • Biological samples include, but are not limited, samples that can provide nucleic acids for analysis.
  • Biological samples include, but are not limited, any derivative or fraction of the aforementioned biological samples.
  • the term “biological sample” is synonymous with the term “nucleic acids.”
  • treatment refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject.
  • “treating” or “treatment” includes ameliorating at least one physical or clinical parameter, which may be indiscernible by the subject.
  • “treating” or “treatment” includes modulating the disease or disorder, either physically or clinically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder.
  • the term “patient” refers to a human male or female subject. The methods and uses of the invention described herein are useful to treat a human. In certain embodiments, the term “subject” refers to a “patient.”
  • cell free nucleic acids refers to the group of nucleic acids, including, but not limited to DNA (cfDNA) and RNA (cfRNA).
  • Cell-free DNA cfDNA
  • cfDNA is DNA circulating freely in bodily fluids such as circulating blood, urine, lymph, interstitial fluid, etc.
  • cfDNA may be extracted from bodily fluids, such as blood, plasma, and urine.
  • cfDNA may be arise from apoptotic cells, necrotic cells, and intact cells that are released into the bloodstream or other bodily fluid and eventually lysed.
  • the cfDNA is a form of cell-free tumor DNA (ctDNA).
  • the cfDNA is referred to synonymously as a cfDNA fragment.
  • single nucleotide variant refers to a substitution in a single nucleotide at a specific position in the genome.
  • nucleotide insertion refers to one or more nucleotides inserted into the genome of a biological sample.
  • deletion refers to one or more nucleotides that has been deleted from genome of a biological sample.
  • translocation refers to the movement in position of one or more nucleotides to another position in the genome.
  • copy number changes refers to a sequence of nucleotides that has been duplicated and placed in the genome.
  • primer extension refers to a technique whereby the 5’ ends of a nucleic acid sequence can be mapped. Primer extension is performed by annealing a specific oligonucleotide primer to a position downstream of a nucleic acid template’s 5’ end. Using a reverse transcriptase, the primer can be extended to create a copy of the template.
  • DNA modifications refers to methylations, hydroxy methylations, and/or strand specific DNA deaminations that may occur to the nucleotides in DNA.
  • a “first target-specific primer” is an oligonucleotide comprising a nucleic acid sequence that can specifically anneal, under suitable annealing conditions, to a target nucleotide sequence of a template nucleic acid. During amplification, the first target- specific primer generates a strand that is complementary to its template, and this complementary strand is capable of being hybridized with a first adapter primer.
  • a “first adapter primer” is an oligonucleotide comprising a nucleic acid sequence that can specifically anneal, under suitable annealing conditions, to a complementary sequence of an adapter nucleic acid.
  • the first adapter primer is therefore identical to at least a portion of the adapter, it anneals to the complementary strand generated by the first target specific-primer to allow amplification to proceed.
  • a “second target-specific primer” is an oligonucleotide comprising a nucleic acid sequence that can specifically anneal, under suitable annealing conditions, to a portion of the target nucleotide sequence comprised by the amplicon resulting from a preceding amplification step. During amplification, the second target-specific primer generates a strand that is complementary to its template, and this complementary strand is capable of being hybridized with a second adapter primer.
  • a “second adapter primer” is an oligonucleotide comprising a nucleic acid sequence that can specifically anneal, under suitable annealing conditions, to a complementary sequence of an adapter nucleic acid.
  • the first adapter primer is therefore identical to at least a portion of the adapter, it anneals to the complementary strand generated by the second target specific-primer to allow amplification to proceed.
  • polymerase extension refers to template-dependent addition of at least one complementary nucleotide, by a nucleic acid polymerase, to the 3' end of a primer that is annealed to a nucleic acid template.
  • nucleic acid adapter or “adapter” refers to a nucleic acid molecule that may be ligated to a nucleic acid comprising a target nucleotide sequence to provide one or more elements useful during amplification and/or sequencing of the target nucleotide sequence.
  • heteromeric is used to describe a positional relationship between the annealing site of a primer of a primer pair and the annealing site of another primer of another primer pair.
  • a second primer is nested by 1, 2, 3 or more nucleotides relative to a first primer, meaning that it binds to a site on the template strand that is frame-shifted by 1, 2, 3 or more nucleotides.
  • nucleic acid polymerase refers to an enzyme that catalyzes the template-dependent polymerization of nucleoside triphosphates to form primer extension products that are complementary to the template nucleic acid sequence.
  • a nucleic acid polymerase enzyme initiates synthesis at the 3’ end of an annealed primer and proceeds in the direction toward the 5’ end of the template.
  • Numerous nucleic acid polymerases are known in the art and are commercially available.
  • One group of nucleic acid polymerases are thermostable, i.e., they retain function after being subjected to temperatures sufficient to denature annealed strands of complementary nucleic acids, e.g., 94°C, or sometimes higher.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, the term “about” indicates the designated value ⁇ one standard deviation of that value.
  • strand separation means treatment of a nucleic acid sample such that complementary double-stranded molecules are separated into two single strands available for annealing to an oligonucleotide primer.
  • strand separation according to methods described herein is achieved by heating the nucleic acid sample above its melting temperature (Tm).
  • anneal refers to the formation of one or more complementary base pairs between two nucleic acids.
  • amplification regimen refers to a process of specifically amplifying (increasing the abundance of) a nucleic acid of interest.
  • substantially anneal refers to an extent to which complementary base pairs form between two nucleic acids that, when used in the context of a PCR amplification regimen, is sufficient to produce a detectable level of a specifically amplified product.
  • a “buffer” may include solvents (e.g., aqueous solvents) plus appropriate cofactors and reagents which affect pH, ionic strength, etc.
  • “primer” refers to an oligonucleotide capable of specifically annealing to a nucleic acid template and providing a 3' end that serves as a substrate for a template- dependent polymerase to produce an extension product which is complementary to the template.
  • next-generation sequencing refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g., Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel.
  • the present disclosure also provides a method for diagnosis by determining whether a DNA has been modified by evaluating DNA modification status, preferably with respect to a single biological sample.
  • the present disclosure also sets forth processes that are advantageous over existing methods to determine the presence of any DNA modifications in a sample. Specifically, the present disclosure sets forth processes that are mutually exclusive with respect to identifying DNA modifications and nucleotide identity from isolated DNA. This thereby preserves the isolated DNA for one or more experiments.
  • DNA methylation analysis may also be performed using enrichment techniques.
  • Affinity enrichment is a technique that is often used to isolate methylated DNA from the rest of the DNA population. This is usually accomplished by antibody immunoprecipitation methods or with methyl-CpG binding domain (MBD) proteins.
  • MBD methyl-CpG binding domain
  • the present disclosure sets forth processes that are important in evaluating both the DNA modification status and nucleotide identity on the same fragment. Evaluating both modification status and nucleotide identity on the same cfDNA advantageously increases the specificity of detection and links possible variants to its expression.
  • the present disclosure sets forth processes that are important for conserving scarce samples of DNA isolated from a subject.
  • the conservation is important because it does not have the disadvantage of splitting a DNA sample for separate evaluations and losing vital materials.
  • cfDNA cell-free DNA
  • every time a sample is split to perform a new experiment the sensitivity of the individual assay is sacrificed.
  • the present disclosure sets forth processes that allow greater efficiency when processing large volumes of samples for genomics studies.
  • cfDNA isolation requires large volumes of samples and purification reagents. These large volumes present operational challenges to processing large numbers of samples in a timely and cost effective manner.
  • the present disclosure allows a user to obtain the same amount, if not more information, from the same or fewer samples.
  • the disclosure relates to the preparation of cfDNA for analysis.
  • preparative techniques described herein may be useful for analysis of cfDNA.
  • cfDNA screening tests can also be used to screen for tumor DNA, for example, as present in the blood of a cancer patient.
  • ctDNA is compared to a patient’s genome providing minimally-invasive cancer diagnosis, prognosis, and tumor monitoring.
  • suitable protocols for the extraction of cfDNA from bodily fluids are used to obtain a cfDNA sample for use in the methods described herein.
  • a suitable protocol for isolation of cfDNA from blood comprises centrifugation of a blood, serum, or plasma sample, followed by isolation and purification of cfDNA from the sample.
  • similar steps are performed for analyzing ctDNA, in which blood are processed.
  • the blood is processed by centrifugation to remove all the cells, while the supernatant is processed to obtain cfDNA.
  • a biological sample comprising methylated DNA is first immunoprecipitated using an antibody to separate methylated DNA from un-methylated DNA.
  • techniques described herein are useful for evaluating tumor DNA and mutation detection.
  • tumor tissue is evaluated to detect cell-free tumor DNA.
  • Cell-free tumor DNA is present in a wide range of cancers but occurs at different levels and mutant allele fractions.
  • ctDNA is highly fragmented to approximately 170 bp.
  • ctDNA molecules are released by tumor cells and circulate in the blood of cancer patients.
  • assays using these molecules are used for early tumor detection, monitoring, or detection of resistance mutations.
  • cell-free fetal DNA is isolated.
  • cffDNA originates in trophoblasts, which may be found in the placenta.
  • NIPT non-invasive prenatal testing
  • the techniques described herein are used for preparing samples of fetal DNA and mutation detection.
  • the techniques described herein are used on studies focused on detecting paternally inherited sequences to detect fetal DNA.
  • primers that have been designed to target the Y chromosome of male fetuses for polymerase chain reaction (PCR) are used.
  • differences in gene activation between maternal DNA and fetal DNA are exploited.
  • epigenetic modifications are made to detect cffDNA.
  • a hypermethylated promoter is used as a universal fetal marker to confirm the presence of cell-free fetal DNA.
  • DNA from the biological sample is amplified to produce an amplified DNA sample.
  • the DNA is amplified by standard techniques such as PCR.
  • the amplified DNA is analyzed for the nucleotide composition.
  • the nucleotide composition is analyzed for the presence of SNV (SNVs), nucleotide insertions or deletions (indels), translocations, and copy number changes, or any combination thereof.
  • one or more rounds of amplification are used.
  • a first round of amplification is conducted using a first target-specific primer and a first adapter primer.
  • a first target-specific primer in the first PCR amplification cycle of the first amplification step, is specifically annealed to a template strand of a nucleic acid comprising a target nucleotide sequence.
  • a sequence upstream or downstream of the target nucleotide sequence is synthesized as a strand complementary to the template strand.
  • the 3' end of the newly synthesized complementary strand comprises a sequence capable of hybridizing with a first adapter primer.
  • both the first target-specific primer and the first adapter primer are able to specifically anneal to the appropriate strands of the target nucleic acid sequence and the sequence between the known nucleotide target sequence and the adapter are amplified.
  • a second round of amplification is conducted using a second target-specific primer and a second adapter primer.
  • a second target-specific primer is nested relative to a first target- specific primer.
  • the use of nested adapter primers eliminates the possibility of producing final amplicons that are amplifiable (e.g., during bridge PCR or emulsion PCR) but cannot be sequenced, a situation that can arise during hemi-nested methods.
  • hemi-nested approaches using a primer identical to a sequencing primer results in the carry-over of undesired amplification products from the first PCR step to the second PCR step and ultimately yields artificial sequencing reads.
  • a second target-specific primer is nested with respect to a first target-specific primer by at least 1 nucleotide, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, a second target-specific primer is nested with respect to a first target-specific primer by about 5 nucleotides to about 10 nucleotides, by about 10 nucleotides to about 15 nucleotides, by about 15 nucleotides to about 20 nucleotides, or by about 20 nucleotides or more.
  • the techniques described herein comprise the use of one or more nested primers.
  • the use of nested primers reduces non-specific binding in PCR products due to the amplification of unexpected primer binding sites.
  • a second target-specific primer comprises a 3' portion that specifically anneals to a target nucleotide sequence and a 5' tail that does not anneal to the target nucleotide sequence.
  • the 5' tail comprises a nucleic acid sequence that is identical to a second sequencing primer.
  • multiple primers e.g., one or more target specific primers and/or one or more adapter primers present in a reaction comprises identical 5' tail sequence portions.
  • a 5' tail comprises a GC-rich sequence. In some embodiments, a 5' tail sequence comprises at least 50% GC content, at least 55% GC content, at least 60% GC content, at least 65% GC content, at least 70% GC content, at least 75% GC content, at least 80% GC content, or higher GC content. In some embodiments, a 5' tail sequence comprises at least 60% GC content. In some embodiments, a 5' tail sequence comprises at least 65% GC content.
  • a second round of amplification comprises a second target- specific primer comprising a 5' tail, a first adapter primer, and an additional primer.
  • the additional primer comprises a 3' portion that is identical to the 5' tail of the second target-specific primer. In some embodiments, the additional primer comprises additional sequences 5' to the hybridization sequence that comprises barcodes, index adapter sequences, or sequencing primer sites. In some embodiments, the additional primer comprises a generic sequencing adapter/index primer.
  • the first and second target-specific primers are substantially complementary to the same strand of the target nucleic acid.
  • the portions of the first and second target-specific primers that specifically anneal to the known target sequence comprises a total of at least 20 unique bases of the known target nucleotide sequence, e.g., 20 or more unique bases, 25 or more unique bases, 30 or more unique bases, 35 or more unique bases, 40 or more unique bases, or 50 or more unique bases.
  • the portions of the first and second target-specific primers that specifically anneal to the known target sequence comprises a total of at least 30 unique bases of the known target nucleotide sequence.
  • the first adapter primer comprises a nucleic acid sequence identical to about the 20 5 '-most bases of the amplification strand of the adapter and the second adapter primer comprises a nucleic acid sequence identical to about 30 bases of the amplification strand of the adapter, with a 5' base that is at least 1 nucleotide 3' of the 5' terminus of the amplification strand.
  • an adapter ligated nucleic acid (e.g., a ligation product) is minimal.
  • a first adapter primer may be used comprises a portion of the adapter nucleic sequence at its 3' end and then additional sequencer-important information at its 5' end.
  • a second adapter primer may be used comprises, at its 3' end, the 5' end of the first adapter primer.
  • the second adapter primer also has a nucleotide sequence that permits sequencing at its 5' end.
  • primers e.g., first and second target-specific primers and first and second adapter primers
  • primers are designed such that they will specifically anneal to their complementary sequences at an annealing temperature of from about 61 to 72°C., e.g., from about 61 to 69°C., from about 63 to 69°C., from about 63 to 67°C., from about 64 to 66°C.
  • primers are designed such that they will specifically anneal to their complementary sequences at an annealing temperature of less than 72°C.
  • primers are designed such that they will specifically anneal to their complementary sequences at an annealing temperature of less than 70°C.
  • primers are designed such that they will specifically anneal to their complementary sequences at an annealing temperature of less than 68°C. In some embodiments, primers are designed such that they will specifically anneal to their complementary sequences at an annealing temperature of about 65°C. In some embodiments, systems provided herein are configured to alter vessel temperature (e.g., by cycling between different temperature ranges) to facilitate primer annealing.
  • the portions of the target-specific primers that specifically anneal to the known target nucleotide sequence will anneal specifically at a temperature of about 61 to 72°C, e.g., from about 61 to 69°C, from about 63 to 69°C, from about 63 to 67°C, from about 64 to 66°C. In some embodiments, the portions of the target-specific primers that specifically anneal to the known target nucleotide sequence will anneal specifically at a temperature of about 65°C in a PCR buffer.
  • methods described herein comprise an extension regimen or step.
  • extension proceeds from one or more hybridized random primers, using the nucleic acid molecules which the primers are hybridized to as templates. Extension steps are described herein.
  • one or more random primers hybridizes to substantially all of the nucleic acids in a sample, many of which may not comprise a target nucleotide sequence. Accordingly, in some embodiments, extension of random primers occurs due to hybridization with templates that do not comprise a target nucleotide sequence.
  • the methods described herein involve a polymerase chain reaction (PCR) amplification regimen, involving one or more amplification cycles.
  • Amplification steps of the methods described herein comprise a PCR amplification regimen, i.e., a set of polymerase chain reaction (PCR) amplification cycles.
  • PCR polymerase chain reaction
  • exponential amplification occurs when products of a previous polymerase extension serve as templates for successive rounds of extension.
  • a PCR amplification regimen according to methods disclosed herein may comprise at least one, and in some cases at least 5 or more iterative cycles.
  • each iterative cycle comprises steps of: 1) strand separation (e.g., thermal denaturation); 2) oligonucleotide primer annealing to template molecules; and 3) nucleic acid polymerase extension of the annealed primers.
  • strand separation e.g., thermal denaturation
  • oligonucleotide primer annealing to template molecules
  • nucleic acid polymerase extension of the annealed primers.
  • conditions and times selected may depend on the length, sequence content, melting temperature, secondary structural features, or other factors relating to the nucleic acid template and/or primers used in the reaction.
  • an amplification regimen according to methods described herein is performed in a thermal cycler, many of which are commercially available.
  • methods described herein can comprise linear amplification.
  • amplification steps performed using nested primers may be performed using linear amplification.
  • amplification may be conducted using nucleic acid sequence-based amplification (NASBA).
  • NASBA nucleic acid sequence-based amplification
  • amplification comprises a T7-mediated NASBA reaction.
  • a nucleic acid extension reaction involves the use of a nucleic acid polymerase.
  • a non-limiting example of a protocol for amplification involves using a polymerase (e.g., PHOENIX TAQ ® , VERASEQ ® ) under the following conditions: 98°C for 30 s, followed by 14-22 cycles comprising melting at 98°C for 10 s, followed by annealing at 68°C for 30 s, followed by extension at 72°C for 3 min, followed by holding of the reaction at 4°C.
  • annealing/extension temperatures may be adjusted to account for differences in salt concentration (e.g., 3°C.
  • slowing the ramp rate e.g., l°C/s, 0.5°C/s, 0.28°C/s, 0.1°C/s or slower, for example, from 98°C to 65°C, improves primer performance and coverage uniformity in highly multiplexed samples.
  • systems provided herein are configured to alter vessel temperature (e.g., by cycling between different temperature ranges, having controlled ramp up or down rates) to facilitate amplification.
  • a nucleic acid polymerase is used under conditions in which the enzyme performs a template-dependent extension.
  • the nucleic acid polymerase is DNA polymerase I, Taq polymerase, PHOENIX TAQ ® , polymerase, PHUSION ® , polymerase, T4 polymerase, T7 polymerase, Klenow fragment, Klenow exo-, phi29 polymerase, AMV reverse transcriptase, M-MuLV reverse transcriptase, HIV-1 reverse transcriptase, VERA SEQ ULTRA ® polymerase, VERASEQ ® HF 2.0 polymerase, EnzScript, or another appropriate polymerase.
  • a nucleic acid polymerase is not a reverse transcriptase.
  • a nucleic acid polymerase acts on a DNA template.
  • the nucleic acid polymerase acts on an RNA template.
  • an extension reaction involves reverse transcription performed on an RNA to produce a complementary DNA molecule (RNA-dependent DNA polymerase activity).
  • a reverse transcriptase is a mouse moloney murine leukemia virus (M-MLV) polymerase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, or another appropriate reverse transcriptase.
  • M-MLV mouse moloney murine leukemia virus
  • a nucleic acid amplification reaction involves cycles including a strand separation step generally involving heating of the reaction mixture.
  • heating to 94°C is sufficient to achieve strand separation.
  • a suitable reaction preparation contains one or more salts (e.g., 1 to 100 mM KC1, 0.1 to 10 mM MgCh), at least one buffering agent (e.g., 1 to 20 mM Tris-HCl), and a carrier (e.g., 0.01 to 0.5% BSA).
  • a non-limiting example of a suitable buffer comprises 50 mM KC1, 10 mM Tris-HCl (pH 8.8 at 25°C), 0.5 to 3 mM MgCh, and 0.1% BSA.
  • a further non-limiting example of a suitable buffer comprises 50 mM KC1, 10 mM Tris-HCl (pH 8.8 at 25°C), 0.5 to 5 mM (e.g., approximately 0.5 mM, approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM) MgCh, and 0.1% BSA.
  • a nucleic acid amplification involves annealing primers to nucleic acid templates having a strands characteristic of a target nucleic acid.
  • a strand of a target nucleic acid can serve as a template nucleic acid.
  • annealing involves two complementary or substantially complementary nucleic acid strands hybridizing together.
  • annealing involves the hybridization of primer to a template such that a primer extension substrate for a template-dependent polymerase enzyme is formed.
  • conditions for annealing e.g., between a primer and nucleic acid template) may vary based of the length and sequence of a primer.
  • conditions for annealing are based upon a Tm (e.g., a calculated Tm) of a primer.
  • an annealing step of an extension regimen involves reducing the temperature following a strand separation step to a temperature based on the Tm (e.g., a calculated Tm) for a primer, for a time sufficient to permit such annealing.
  • a Tm can be determined using any of a number of algorithms (e.g., OLIGO ® , (Molecular Biology Insights Inc. Colorado) primer design software and VENTRO NTI ® , (Invitrogen, Inc.
  • the Tm of a primer can be calculated using the following formula, which is used by NetPrimer software and is described in more detail in Frieir, et al. PNAS 1986 83:9373-9377 which is incorporated by reference herein in its entirety.
  • Tm AH/(AS+R*ln(C/4))+16.6 log([K+]/(l+0.7[K+]))-273.15
  • DH enthalpy for helix formation
  • AS entropy for helix formation
  • R molar gas constant (1.987 cal/°C*mol)
  • C is the nucleic acid concentration
  • [K+] salt concentration
  • the annealing temperature is selected to be about 5°C below the predicted Tm, although temperatures closer to and above the Tm (e.g., between 1°C and 5°C below the predicted Tm or between 1°C and 5°C above the predicted Tm) can be used, as can, for example, temperatures more than 5°C below the predicted Tm (e.g., 6°C below, 8°C below, 10°C below or lower). In some embodiments, the closer an annealing temperature is to the Tm, the more specific is the annealing.
  • the time used for primer annealing during an extension reaction is determined based, at least in part, upon the volume of the reaction (e.g., with larger volumes involving longer times). In some embodiments, the time used for primer annealing during an extension reaction (e.g., within the context of a PCR amplification regimen) is determined based, at least in part, upon primer and template concentrations (e.g., with higher relative concentrations of primer to template involving less time than lower relative concentrations).
  • primer annealing steps in an extension reaction can be in the range of 1 second to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 2 minutes.
  • polymerase extension adds more than one nucleotide, e.g., up to and including nucleotides corresponding to the full length of the template.
  • conditions for polymerase extension are based, at least in part, on the identity of the polymerase used.
  • the temperature used for polymerase extension is based upon the known activity properties of the enzyme. In some embodiments, in which annealing temperatures are below the optimal temperatures for the enzyme, it may be acceptable to use a lower extension temperature. In some embodiments, enzymes may retain at least partial activity below their optimal extension temperatures.
  • a polymerase extension (e.g., performed with thermostable polymerases such as Taq polymerase and variants thereof) is performed at 65°C to 75°C, or 68°C to 72°C.
  • methods provided herein involve polymerase extension of primers that are annealed to nucleic acid templates at each cycle of a PCR amplification regimen.
  • a polymerase extension is performed using a polymerase that has relatively strong strand displacement activity.
  • polymerases having strong strand displacement are useful for preparing nucleic acids for purposes of detecting fusions (e.g., 5' fusions).
  • primer extension is performed under conditions that permit the extension of annealed oligonucleotide primers.
  • conditions that permit the extension of an annealed oligonucleotide such that extension products are generated refers to the set of conditions (e.g., temperature, salt and co-factor concentrations, pH, and enzyme concentration) under which a nucleic acid polymerase catalyzes primer extension. In some embodiments, such conditions are based, at least in part, on the nucleic acid polymerase being used.
  • a polymerase may perform a primer extension reaction in a suitable reaction preparation.
  • a suitable reaction preparation contains one or more salts (e.g., 1 to 100 mM KC1, 0.1 to 10 mM MgC12), at least one buffering agent (e.g., 1 to 20 mM Tris-HCl), a carrier (e.g., 0.01 to 0.5% BSA), and one or more NTPs (e.g., 10 to 200 mM of each of dATP, dTTP, dCTP, and dGTP).
  • salts e.g., 1 to 100 mM KC1, 0.1 to 10 mM MgC12
  • buffering agent e.g., 1 to 20 mM Tris-HCl
  • a carrier e.g., 0.01 to 0.5% BSA
  • NTPs e.g., 10 to 200 mM of each of dATP, dTTP, dCTP, and dGTP.
  • a non-limiting set of conditions is 50 mM KC1, 10 mM Tris-HCl (pH 8.8 at 25°C), 0.5 to 3 mM MgCh, 200 mM each dNTP, and 0.1% BSA at 72°C, under which a polymerase (e.g., Taq polymerase) catalyzes primer extension.
  • a polymerase e.g., Taq polymerase
  • a suitable reaction preparation contains one or more salts (e.g., 1 to 100 mM KC1, 0.5 to 5 mM MgC12), at least one buffering agent (e.g., 1 to 20 mM Tris-HCl), a carrier (e.g., 0.01 to 0.5% BSA), and one or more NTPs (e.g, 50 to 350 mM of each of dATP, dTTP, dCTP, and dGTP).
  • salts e.g., 1 to 100 mM KC1, 0.5 to 5 mM MgC12
  • buffering agent e.g., 1 to 20 mM Tris-HCl
  • a carrier e.g., 0.01 to 0.5% BSA
  • NTPs e.g, 50 to 350 mM of each of dATP, dTTP, dCTP, and dGTP.
  • a non-limiting set of conditions is 50 mM KC1, 10 mM Tris-HCl (pH 8.8 at 25°C), 3 mM MgCh, 200 mM each dNTP, and 0.1% BSA at 72°C, under which a polymerase (e.g., Taq polymerase) catalyzes primer extension.
  • a polymerase e.g., Taq polymerase
  • a further non-limiting set of conditions is 50 mM KC1, 10 mM Tris-HCl (pH 8.8 at 25°C), 3 mM MgCh, 266 mM dATP, 200 mM dCTP, 133 mM dGTP, 200 mM dTTP, and 0.1% BSA at 72°C, under which a polymerase (e.g., Taq polymerase) catalyzes primer extension.
  • a polymerase e.g., Taq polymerase
  • conditions for initiation and extension may include the presence of one, two, three or four different deoxyribonucleoside triphosphates (e.g., selected from dATP, dTTP, dCTP, and dGTP) and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer.
  • the two, three or four different deoxyribonucleoside triphosphates are present in equimolar, or approximately equimolar, concentrations.
  • the two, three or four different deoxyribonucleoside triphosphates are present in different concentrations, which have been experimentally determined to be suitable to a particular implementation of the technology.
  • nucleic acid amplification involves up to 5, up to 10, up to 20, up to 30, up to 40 or more rounds (cycles) of amplification.
  • nucleic acid amplification may comprise a set of cycles of a PCR amplification regimen from 5 cycles to 20 cycles in length.
  • an amplification step may comprise a set of cycles of a PCR amplification regimen from 10 cycles to 20 cycles in length.
  • each amplification step can comprise a set of cycles of a PCR amplification regimen from 12 cycles to 16 cycles in length.
  • an annealing temperature can be less than 70°C.
  • an annealing temperature can be less than 72°C.
  • an annealing temperature can be about 65°C.
  • an annealing temperature can be from about 61 to about 72°C.
  • methods and compositions described herein relate to performing a PCR amplification regimen with one or more of the types of primers described herein.
  • a primer is single-stranded, such that the primer and its complement can anneal to form two strands.
  • Primers according to methods and compositions described herein may comprise a hybridization sequence (e.g., a sequence that anneals with a nucleic acid template) that is less than or equal to 300 nucleotides in length, e.g., less than or equal to 300, or 250, or 200, or 150, or 100, or 90, or 80, or 70, or 60, or 50, or 40, or 30 or fewer, or 20 or fewer, or 15 or fewer, but at least 6 nucleotides in length.
  • a hybridization sequence of a primer may be 6 to 50 nucleotides in length, 6 to 35 nucleotides in length, 6 to 20 nucleotides in length, 10 to 25 nucleotides in length.
  • Any suitable method may be used for synthesizing oligonucleotides and primers.
  • commercial sources offer oligonucleotide synthesis services suitable for providing primers for use in methods and compositions described herein (e.g., Invitrogen, Custom DNA Oligos (Life Technologies, Grand Island, N. Y.) or custom DNA Oligos from Integrated DNA Technologies (Coralville, Iowa)).
  • the isolated cfDNA is purified from other enzymes, primers, or buffer components using any appropriate step or method.
  • the isolated cfDNA is purified using a suitable commercially available kit.
  • amplification products are isolated from enzymes, primers, or buffer components before any appropriate step of a method.
  • any suitable method for isolating nucleic acids may be used.
  • the isolation comprises Solid Phase Reversible Immobilization (SPRI) cleanup.
  • SPRI cleanup are well known in the art, e.g., Agencourt AMPure XP-PCR Purification (Cat No. A63880, Beckman Coulter; Brea, Calif.).
  • enzymes are inactivated by heat treatment.
  • unlabeled dNTPs are removed by enzymatic treatment.
  • unhybridized primers are removed from a nucleic acid preparation using appropriate methods (e.g., purification, digestion, etc.).
  • a nuclease e.g., exonuclease I
  • such nucleases are heat inactivated subsequent to primer digestion.
  • a further set of primers may be added together with other appropriate components (e.g., enzymes, buffers) to perform a further amplification reaction.
  • steps of the methods provided herein optionally comprise an intervening sample purification step.
  • a sample purification step comprises a wash step.
  • a sample purification step comprises SPRI cleanup (e.g., AMPure).
  • the purified cfDNA is sheared (e.g., mechanically or enzymatically sheared, sheared via nebulizer) to generate fragments of any desired size.
  • sheared e.g., mechanically or enzymatically sheared, sheared via nebulizer
  • mechanical shearing processes include sonication, nebulization, and AFA ® shearing technology available from Covaris (Woburn,
  • a nucleic acid are mechanically sheared by sonication.
  • a target nucleic acid is not sheared or digested.
  • nucleic acid products of preparative steps e.g., extension products, amplification products are not sheared or enzymatically digested.
  • the methods comprise binding the cfDNA with an adapter.
  • an adapter is single-stranded.
  • an adapter is double-stranded.
  • a double-stranded adapter comprises a first ligatable duplex end and a second unpaired end.
  • an adapter comprises an amplification strand and a blocking strand.
  • the amplification strand comprises a 5' unpaired portion and a 3' duplex portion.
  • the amplification strand further comprises a 3' overhang.
  • the 3' overhang is a 3' T overhang.
  • the amplification strand comprises nucleotide sequences identical to a first and second adapter primer.
  • the blocking strand of the adapter comprises a 5' duplex portion and a non-extendable 3' portion.
  • the blocking strand further comprises a 3' unpaired portion.
  • the duplex portions of the amplification strand and the blocking strand are substantially complementary and the duplex portion is of sufficient length to remain in duplex form at the ligation temperature.
  • the portion of the amplification strand that comprises a nucleotide sequence identical to a first and second adapter primer can be comprised, at least in part, by the 5' unpaired portion of the amplification strand.
  • the adapter comprises a “Y” shape, i.e., the second unpaired end comprises a 5' unpaired portion of an amplification strand and a 3' portion of a blocking strand.
  • the 3' unpaired portion of the blocking strand is shorter than, longer than, or equal in length to the 5' unpaired portion of the amplification strand.
  • the 3' unpaired portion of the blocking strand is shorter than the 5' unpaired portion of the amplification strand.
  • Y- shaped adapters have the advantage that the unpaired portion of the blocking strand will not be subject to 3' extension during a PCR regimen.
  • compositions that can be used to identify or analyze nucleic acids.
  • the composition comprises a pool of Y-shaped adaptors, wherein each Y-shaped adaptor comprises a hybridizable portion at one end of the Y-shaped adaptor and a non-hybridizable portion at the opposite end of the Y-shaped adaptor, wherein the hybridizable portion comprises a unique identifiable double-stranded stem barcode of at least two base pairs.
  • compositions comprising a pool of Y-shaped adaptors, wherein each Y-shaped adaptor comprises a hybridizable portion at one end of the Y-shaped adaptor and a non-hybridizable portion at the opposite end of the Y-shaped adaptor, wherein the non-hybridizable portion comprises i) a pre-defmed single-stranded barcode of at least two nucleotides, and ii) a random single-stranded barcode of at least two nucleotides on the same strand as the pre-defmed single-stranded barcode.
  • compositions that can include a pool of Y-shaped adaptors, wherein each Y-shaped adaptor comprises a hybridizable portion at one end of the Y-shaped adaptor and a non-hybridizable portion at the opposite end of the Y-shaped adaptor, wherein the hybridizable portion comprises a unique double-stranded stem barcode of at least two nucleotides, and wherein the non-hybridizable portion comprises i) a pre-defmed single-stranded barcode of at least two nucleotides, and ii) a random single-stranded barcode of at least two nucleotides on the same strand as the pre-defmed single-stranded barcode.
  • the adaptors comprise a pre-defmed single-stranded barcode and a random single-stranded barcode on the 5' strand of the non-hybridizable portion of the adaptor.
  • the pre-defmed single-stranded barcode and the random single-stranded barcode is on the 3' strand of the non-hybridizable portion of the adaptor.
  • the pre-defmed single-stranded barcode is adjacent to the random single-stranded barcode. It is also explicitly contemplated that the pre-defmed single- stranded barcode can be separated from the random single-stranded barcode by one or more nucleotides.
  • the pre-defmed single-stranded barcode comprises naturally occurring bases ⁇ e.g., Adenosine (A), Thymidine (T), Guanosine (G), Cytosine (C), and Uracil (U)) or non-naturally occurring bases e.g., aminoallyl-uridine, iso-cytosines, isoguanine, and 2- aminopurine, and be between 1 and about 20 nucleotides long.
  • naturally occurring bases e.g., Adenosine (A), Thymidine (T), Guanosine (G), Cytosine (C), and Uracil (U)
  • non-naturally occurring bases e.g., aminoallyl-uridine, iso-cytosines, isoguanine, and 2- aminopurine, and be between 1 and about 20 nucleotides long.
  • the length of the random barcode comprises between 1 and about 20 nucleotides and it can contain naturally occurring bases ⁇ e.g., Adenosine (A), Thymidine (T), Guanosine (G), Cytosine (C), and Uracil (U)), or non-naturally occurring bases e.g., aminoallyl-uridine, iso-cytosines, isoguanine.
  • naturally occurring bases e.g., Adenosine (A), Thymidine (T), Guanosine (G), Cytosine (C), and Uracil (U)
  • non-naturally occurring bases e.g., aminoallyl-uridine, iso-cytosines, isoguanine.
  • the length of the double-stranded stem barcode comprises between 1 and about 20 nucleotides.
  • the double-stranded stem barcode comprises a pre-defmed sequence. In certain embodiments, the double-stranded stem barcode comprises random sequence or comprise both a pre-defmed sequence and a random sequence.
  • the double-stranded barcode comprises natural and non-natural nucleotides, e.g., aminoallyl-uridine, iso-cytosines, isoguanine, and 2-aminopurine, which assists in the detection of the double-stranded barcode.
  • natural and non-natural nucleotides e.g., aminoallyl-uridine, iso-cytosines, isoguanine, and 2-aminopurine, which assists in the detection of the double-stranded barcode.
  • each Y-shaped adaptor comprises a primer sequence.
  • the primer sequence is a PCR primer sequence or a sequencing primer sequence. In some embodiments, the primer sequence is on the non-hybridizable portion of the Y-shaped adaptor.
  • the primer sequence is on the hybridizable portion of the Y-shaped adaptor. In some embodiments, the primer sequence is the same in the entire Y-shaped adaptor pool. In some other embodiments, the primer sequences on one or more Y-shaped adaptors is different from the primer sequences on other Y-shaped adaptors.
  • the blocking strand of the adapter comprises a 3' unpaired portion that is not substantially complementary to the 5' unpaired portion of the amplification strand, wherein the 3' unpaired portion of the blocking strand is not substantially complementary to or substantially identical to any of the primers.
  • the blocking strand comprises a 3' unpaired portion that does not specifically anneal to the 5' unpaired portion of the amplification strand at the annealing temperature, wherein the 3' unpaired portion of the blocking strand will not specifically anneal to any of the primers or the complements thereof at the annealing temperature.
  • an adapter nucleic acid comprises, at a minimum, a sample index sequence for multiplexing. In certain embodiments, the adapter nucleic comprises a random molecular barcode.
  • the adaptors disclosed herein and their specific embodiments can be attached to the one or more nucleic acids through the hybridizable (double-stranded) portion of the adaptors.
  • the adaptors can have free or linked single stranded portions.
  • the method utilizes adaptors with free single stranded portions (Y-shaped adaptors) and covalently linked single-stranded portions (BAL-Seq adaptors) or a combination of two types of adaptors.
  • the covalently linked single-stranded portions are linked by a linker.
  • the linker may optionally contain a cleavage site, e.g., a restriction enzyme recognition sequence.
  • the adaptors have barcodes located according to several distinct embodiments described below.
  • the adaptors have one or more barcodes on each single-stranded portion and one or more barcodes in the double stranded portion.
  • each of the barcodes is located (or co-located) in (a) upper single stranded region (containing the 5'-end), (b) lower single stranded region (containing the 3'-end), and (c) the double-stranded region or stem of the Y-shaped adaptor.
  • the method utilizes unique molecular identifiers (UMIs).
  • UMIs are capable of ligating to the ends of the DNA fragment.
  • the UMI is present adjacent to the sample index position.
  • the UMI is between and including 3-10 nucleotides.
  • the UMI is between and including 3-9 nucleotides.
  • the UMI is between and including 3-8 nucleotides.
  • the UMI is between and including 3-7 nucleotides.
  • the UMI is between and including 3-6 nucleotides.
  • the UMI is between and including 3-5 nucleotides.
  • the UMI is between and including 3-4 nucleotides.
  • the UMIs are on both strands of the adaptor: the upper and the lower strands, or in the double stranded region. In some embodiments, if the UMIs are matched as originating from the same adaptor, double strand sequencing (i.e., pairing single strands is possible) is used. In some embodiments, the UMIs located in the double stranded region are matched by Watson-Crick pairing. In some embodiments, the known-sequence (not random) UMIs present on the single stranded portions are cross-referenced as belonging to the same adaptor molecule.
  • the random single-stranded barcode combined with an endogenous barcode provide a unique identifier for each template nucleic acid.
  • the endogenous barcode comprises a sequence of any length and comprises one or more sets of nucleotide sequences on a nucleic acid. In certain embodiments, the sequences are at different loci of the nucleic acid.
  • the endogenous barcode comprises a first sequence on an end of the nucleic acid and a second sequence on the opposite end of the nucleic acid.
  • the endogenous barcode comprises an internal sequence. In certain embodiments, the endogenous barcode comprises a first sequence that is internal, and a second sequence that is on one end of the nucleic acid. In some embodiments, the endogenous barcode comprises a first and a second sequence that are both internal.
  • the amplicons derived from the same template nucleic acid contain the same UMIs. These distinct unique identifiers are used to identify and count the distinct template nucleic acids in the original sample. For example, UMIs can be used to count original template nucleic acids containing the same mutations. In some embodiments, UMIs are used to identify and group the amplicons from the same original template nucleic acid.
  • the stem barcode is in any portion of the stem of the adaptor.
  • the stem barcode is adjacent to the base pair to which the adaptor attaches on the nucleic acid or one or more base pairs away from the base pair to which the adaptor attaches on the nucleic acid.
  • the unique double-stranded stem barcodes identify strands of the nucleic acid. For example, after an adaptor is attached to a nucleic acid, both strands of the resulting nucleic acid contain the unique stem barcode, even though each strand of the nucleic acid may contain different random single-stranded barcodes or different unique identifier. After amplification, the amplicons derived from one strand of the nucleic acid contain the same stem barcode and the same endogenous barcode as the amplicons derived from the other strand of the same nucleic acid. In some embodiments, the stem barcode identifies amplicons derived from the two strands of the same template nucleic acid.
  • the unique stem barcodes identify mutations on one strand, but not the other strand, of the nucleic acid. In some other embodiments, mutations that occur on one strand, but not the other strand, of the template nucleic acid are the result of amplification errors and are disregarded as artifact.
  • the method comprises using “tandem” sequencing adaptors containing two fundamentally distinct barcodes, which allow tracking of individual DNA molecules to distinguish real somatic mutations arising in vivo from errors introduced during ex vivo procedures including high-throughput sequencing.
  • adaptors comprise barcodes that include a defined sequence or a random sequence or a combination of a random sequence and a defined sequence.
  • the single stranded portion of the adaptor includes a barcode consisting of a multiplex sample ID (MID) portion shared among the adaptor molecules in a sample and a barcode unique to each adaptor molecule.
  • MID multiplex sample ID
  • the unique barcode is a random barcode.
  • Adaptors with such compound barcodes are referred to as “index adaptors.”
  • a typical sample multiplexing barcode (MID) is replaced with a degenerate UMI.
  • a short UMI (2 or more nucleotides) near the ligating end of the adaptor creates an “insert” or internal barcode or internal UMI.
  • the cfDNA is labeled with an adapter prior to any amplification.
  • the adapter linked cfDNA is separated from amplified DNA.
  • this separation can be performed by chromatography or affinity tag techniques.
  • the adapter linked biological DNA is subjected to additional analysis of nucleotide composition.
  • the adapter linked DNA is analyzed for the presence of SNV, nucleotide insertions or deletions, translocations, or copy number changes, or any combination thereof.
  • the method comprises tagging the nucleic acid sample by chemically ligating a tag to the DNA.
  • this tag is known as a capture moiety.
  • the tag comprises: biotin, dual biotin, fluorescently modified bases, alkyne modified base, functional groups allowing orthogonal chemistry, digoxigenin modified base, purification tags, and unique molecular identifiers (UMIs), or any combination thereof.
  • nucleic acid end repair is performed on the cfDNA or amplified products.
  • the end repair reaction is conducted prior to attaching the adaptors to cfDNA.
  • the end repair reaction is conducted after amplification of the adaptor-modified nucleic acids.
  • the end repair reaction is conducted prior to fragmenting the DNA. In other embodiments, the end repair reaction is conducted after fragmenting the DNA. [00131] In some embodiments, the end repair reaction is performed by using one or more end repair enzymes.
  • enzymes for repairing DNA comprise polymerase and exonuclease.
  • a polymerase can fill in the missing bases for a DNA strand from 5' to 3' direction.
  • the resulting double-stranded DNA can be the same length as the original longest DNA strand.
  • An Exonuclease can remove the 3' overhangs.
  • the resulting double-stranded DNA can be the same length as the original shortest DNA strand.
  • the method comprises performing an A-tailing reaction on the cfDNA to produce a cfDNA with an A-tail.
  • the A-tailing reaction is conducted prior to attaching the adaptors to the cfDNA.
  • the A-tailing reaction is conducted prior to fragmenting the cfDNA.
  • the A-tailing reaction is conducted prior to end repair of the cfDNA.
  • the A-tailing reaction is performed by using one or more A- tailing enzymes.
  • an A residue is added by incubating a DNA fragment with dATP and a non-proofreading DNA polymerase, which adds a single 3’ A residue.
  • the ligated material is immobilized on solid matrix.
  • the ligated materials are immobilized on a solid matrix by magnetic beads, bio- streptavidin, click chemistry, antibody-dioxigenin, or any combination thereof.
  • the method comprises producing a copied DNA fragment from the immobilized tagged sample. In certain embodiments, the method comprises evaluating the DNA mutation status of the DNA fragment in the sample from the copied DNA fragment. In certain embodiments, the DNA mutation status provides information on the DNA methylation status.
  • each strand of the DNA fragments in the tagged sample is bound separately to the substrate.
  • a binding partner is attached to an insoluble support.
  • the molecule of interest may be immobilized on an insoluble support through a selective binding interaction formed between a capture moiety, present on the adapter, and a binding partner of the capture moiety attached to the insoluble support.
  • the insoluble support comprises a bead or other solid surface.
  • the bead is a paramagnetic bead.
  • the use of beads for isolation is well known in the art, and any suitable bead isolation method can be used with the techniques described herein.
  • beads that are useful for isolation of the molecules of interest are attached to the beads, and the beads are washed to remove solution components not attached to the beads, allowing for purification and isolation.
  • the beads are separated from other components in the solution based on properties such as size, density, or dielectric, ionic, and magnetic properties.
  • the insoluble support is a magnetic bead. Use of beads allows the derivatized nucleic acid capture moiety to be separated from a reaction mixture by centrifugation or filtration, or, in the case of magnetic beads, by application of a magnetic field.
  • magnetic beads can be introduced, mixed, removed, and released into solution using magnetic fields.
  • processes utilizing magnetic beads is automated.
  • the beads are functionalized using well known chemistry to provide a surface having suitable functionalization for attaching a binding partner of a capture moiety. Derivatization of surfaces to allow binding of the capture moiety is conventional in the art. For example, coating of surfaces with streptavidin allows binding of a biotinylated capture moiety. Coating of surfaces with streptavidin has been described in, for example, U.S. Pat. No. 5,374,524 to Miller.
  • solid surfaces other than beads may be used. In some embodiments, the solid surfaces are planar surfaces, such as those used for hybridization microarrays, or the solid surfaces are the packing of a separation column.
  • a binding partner of a capture moiety may be attached to an insoluble support before, simultaneous with, or after binding the capture moiety.
  • the capture moiety is contacted with a binding partner of the capture moiety while both are in solution.
  • the capture moiety: binding partner complex is immobilized on an insoluble support by contacting the complex with an appropriately derivatized surface.
  • the molecule of interest are isolated through a complex formed between a capture moiety attached to the molecule of interest and a binding partner of the capture moiety.
  • the tagged biological DNA remains in its native state until such a time that its sequence composition is altered.
  • the DNA is analyzed for chemical modifications.
  • the modification is methylation or hydroxymethylation of cytosines at CpG dinucleotides.
  • the modification is stand deamination or the presence of N-methyladenine bases.
  • the tagged biological DNA is analyzed for methylation.
  • the methylation is analyzed by bisulfite conversion.
  • the methylation is analyzed by enzymatic deamination.
  • the methylation is analyzed by selective enriched based on the presence or absence of methylation.
  • the methylation is analyzed by TET-assisted pyridine borane sequencing (TAPS).
  • TAPS TET-assisted pyridine borane sequencing
  • the bisulfite conversion analysis is able to obtain information on the nucleotide composition and modification status on the exact nucleotide.
  • methylated DNA residues comprises bisulfite sequencing and enzymatic based methods of DNA based methods of non-methylated Cytosine conversion to Uracil.
  • the DNA is processed for identification of methylated or hydroxymethylated C residues by using bisulfite treatment.
  • the DNA is processed by enzymatic deamination of unmethylated C residues via APOBEC following treatment of DNA with TET2 enzyme, to produce restriction enzyme cleavage of fragments with unmethylated restriction sites present.
  • TET2 enzyme a restriction enzyme cleavage of fragments with unmethylated restriction sites present.
  • the DNA is analyzed for chemical modifications.
  • the modification is methylation or hydroxymethylation of cytosines at CpG dinucleotides.
  • the modification is strand deamination or the presence of N-methyladenine bases.
  • the DNA is analyzed for its composition and chemical modifications.
  • the method comprises the cconcurrent analysis of DNA methylation status and the identification of single nucleotide variations.
  • the method comprises the concurrent analysis of DNA methylation status and the identification of insertions and/or deletions in cfDNA.
  • the evaluation of DNA mutation status identifies single nucleotide variations.
  • the method comprises performing primer extension on the sample.
  • the primer extension products are subjected to subsequent analysis of DNA modification status.
  • the method of any of the previous claims, wherein the bound biological DNA can be processed for analysis of the DNA modification status.
  • the technology described herein relates to methods of DNA sequencing.
  • the sequencing is performed by a next-generation sequencing method.
  • next-generation sequencing methods/platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro-sequencing (454 Life Sciences/Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexa/Illumina); SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, and Oxford Nanopore Technologies.
  • the sequencing primers comprise portions compatible with the selected next-generation sequencing method.
  • the sequencing step relies upon the use of a first and second sequencing primer.
  • the first and second sequencing primers are selected to be compatible with a next-generation sequencing method as described herein.
  • methods of aligning sequencing reads to known sequence databases of genomic and/or cDNA sequences are well known in the art, and software is commercially available for this process.
  • reads (less the sequencing primer and/or adapter nucleotide sequence) which do not map, in their entirety, to wild-type sequence databases are genomic rearrangements or large insertions or deletion mutations.
  • reads (less the sequencing primer and/or adapter nucleotide sequence) comprising sequences which map to multiple locations in the genome are genomic rearrangements.
  • a de novo assembly of reads overlapping into contiguous sequences, or “contigs,” are built and utilized in the alignment of sequencing reads.
  • a hot spot reference is utilized that does not rely on a publicly accessible genomics database.
  • genotyping, detection, identification, or quantitation of the ctDNA utilizes sequencing.
  • sequencing is accomplished using high- throughput systems.
  • sequencing is performed using the cfDNA described herewithin.
  • sequence information of the cfDNA sample is obtained by massively parallel sequencing.
  • massively parallel sequencing is performed on a subset of a genome, e.g., from a subset of cfDNA from the cfDNA sample.
  • sequence information is obtained by parallel sequencing using flow cells.
  • primers for amplification are covalently attached to slides in the flow cells and then the flow cells are exposed to reagents for nucleic acids extension and sequencing.
  • high-throughput sequencing utilizes the technology available from Helicos Biosciences Corp. (Cambridge, Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS) method.
  • high-throughput sequencing involves the use of technology available by 454 Life Sciences, Inc. (Branford, Conn.) such as the Pico Titer Plate device which includes a fiber optic plate that transmits chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. This use of fiber optics allows for the detection of a minimum of 20 million base pairs in 4.5 hours.
  • the high-throughput sequencing utilizes next generation sequencing techniques, e.g., using the HiSeq or MiSeq instruments available from Illumina (San Diego, Cal.)
  • This sequencing method is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers.
  • the sequencing involves a library preparation step. Genomic DNA is fragmented, and sheared ends are repaired and adenylated. Adaptors are added to the 5' and 3' ends of the fragments. The fragments are size selected and purified.
  • the sequencing comprises a cluster generation step. DNA fragments are attached to the surface of flow cell channels by hybridizing to a lawn of oligonucleotides attached to the surface of the flow cell channel.
  • the fragments are extended and clonally amplified through bridge amplification to generate unique clusters.
  • the fragments become double stranded, and the double stranded molecules can be denatured.
  • Multiple cycles of the solid-phase amplification followed by denaturation creates several million clusters of approximately 1,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Reverse strands are cleaved and washed away. Ends are blocked, and primers hybridize to DNA templates. Hundreds of millions of clusters are sequenced simultaneously.
  • Primers, DNA polymerase, and four fluorophore-labeled, reversible terminator nucleotides are used to perform sequential sequencing. All four bases compete with each other for the template.
  • a laser is used to excite the fluorophores, and an image is captured and the identity of the first base is recorded.
  • the 3' terminators and fluorophores from each incorporated base are removed and the incorporation, detection and identification steps are repeated.
  • a single base can be read each cycle.
  • a HiSeq system ⁇ e.g., HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000) is used for sequencing.
  • high-throughput sequencing of cfDNA takes place using AnyDot- chips (Genovoxx, Germany), which allows monitoring of biological processes (e.g., miRNA expression or allele variability (SNP detection)).
  • AnyDot-chips allow for 10X -50X enhancement of nucleotide fluorescence signal detection.
  • Other high- throughput sequencing systems include those disclosed in Venter, J., et al. Science 16 February 2001; Adams, M. et al, Science 24 March 2000; and M. J, Levene, et al. Science 299:682-686, January 2003; as well as U.S. Application Pub. No. 2003/0044781 and 2006/0078937.
  • the methods disclosed herein comprise conducting a sequencing reaction based on one or more genomic regions from a selector.
  • the sequencing information is obtained for a subset of genomic regions from a selector.
  • sequencing information may be obtained for 10 - 500 or more genomic regions from a selector.
  • sequencing information is obtained for less than 5%, or up to 95% of the genomic regions from a selector. DISEASES
  • the diseases that may be analyzed by the methods herein include, but are not limited to, those disease that have changes in the DNA methylation of parts or all of a genome.
  • the diseases that may be analyzed by this method include, but are not limited to, cancer.
  • the cancers that may be analyzed include, but are not limited to, ovarian, breast, gastric, colon, endometrial, lung, head, neck, colorectal, esophageal, prostate, uterine, pancreatic, kidney, and lymphomas.
  • the diseases that may be analyzed include but are not limited to, various neurological conditions.
  • the neurological conditions that may be analyzed include, but are not limited to Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
  • the methods described herein to treat various diseases provide data on methylation patterns to a medical professional, who in turn decides on a specific course of treatment.
  • the treatment may include, but is not limited to, antibody therapies, small molecule therapies, radiation therapies, and cellular therapies.
  • antibody therapies small molecule therapies
  • radiation therapies and cellular therapies.
  • the nature of treatment will be understood to those of skill in the art, especially as treatments and therapies advance.
  • the methods described herein may be used by a medical professional to treat various diseases by combining any of the previous embodiments with known treatments and methods of chemical modification analysis.
  • the methods described herein may be used by a medical professional for cancer recurrence testing, treatment response monitoring, and asymptomatic early detection, or any combination thereof.
  • the methods described herein may be used by a medical professional to decide to not to administer a treatment. In certain embodiments, the methods herein may be used by a medical professional to decide to stop administering a treatment.
  • the following non-limiting methods are provided to further illustrate the embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of several embodiments of the invention, and thus be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and the scope of the invention.
  • FIG. 1 Exemplary Protocol for Methylation Analysis
  • Figures 1-2 depict exemplary protocols. Each Figure number represents a successive step in the exemplary protocols and are described in the Figure legends.
  • the labeled biological DNA was captured using an affinity matrix, in this case a streptavidin coated magnetic bead.
  • Amplified enzymatic DNA is not bound to the affinity matrix and its sequence composition can be determined by currently available methods or technologies.
  • Bead bound DNA which apart from minute amounts of non-specifically adhered material, is otherwise entirely comprised of the original biological DNA.
  • This DNA carried epigenetic methylation marks, the locations of which were determined by subjecting the bead bound DNA to enzymatic conversion of unmethylated Cytosine to Uracil. Since methylated Cytosines are protected from this conversion, the methylation status was determined by reading Cytosine residues in DNA as such in subsequent analyses.
  • Method 2 Molecular Analysis
  • the methylation status of a region of the BRCA1 promoter was measured in two cell lines, SW620 and OVCAR8.
  • the SW620 cell line lack detectable methylation in a specific region (Clovis Oncology- Personal communication), while in the OVCAR8 cell line, 2 of the same 3 BRCA1 loci are methylated (Kondrashova et al, 2018).
  • the methylation status of the BRCA1 promoter region was assessed in these samples following enzymatic conversion of non-methylated Cytosines to Uracil using hydrolysis probe-based allele specific PCR (Swisher et al, 2021). Approximately 66% methylated BRCA1 promoter in OVCAR8 cells and 0% methylated BRCA1 promoter in SW620 was expected to be observed in cells if conversion is efficient.
  • DNA from the two cell lines was fragmented by sonication using a Covaris LE220 instrument to an average size of 240bp. Following fragmentation, 50 ng of DNA from these cell lines was end-repaired and dA tailed using conventional methods, i.e. T4 DNA polymerase and T4 polynucleotide kinase for end-repair and Taq DNA polymerase for addition of the single dA base 3' overhang (Agilent Technologies). End-repaired and dA tailed DNA was then ligated to a custom double stranded DNA adapter (Integrated DNA Technologies).
  • T4 DNA polymerase and T4 polynucleotide kinase for end-repair
  • Taq DNA polymerase for addition of the single dA base 3' overhang
  • This adapter was synthesized such that every Cytosine residue in the adapter was methylated and the 5' end of the adapter strand distal to the ligation site carried a 5' biotin modification.
  • the ligated product was purified using SPRI beads (Beckman Coulter) to remove free unligated adapter and the other non-DNA reaction components.
  • Purified ligation product was amplified with 6 cycles of PCR using primers that added sample identification indices and sequencing adapters for an Illumina sequencing machine (Agilent Technologies). The original biological DNA was recovered from the product of the PCR by binding to Streptavidin coated magnetic beads (ThermoFisher Scientific).
  • the unbound material was separated from the bead bound material, purified using SPRI beads, and quantified using Quant-it reagent dsDNA assay kit (ThermoFisher Scientific).
  • the unbound material represents amplification product of the biological DNA and carries no epigenetic marks.
  • Bead bound DNA was washed with Hybridization capture wash solutions according to manufacturer recommendations (Integrated DNA Technologies) then subjected to enzymatic Cytosine conversion using a slightly modified workflow of the NEBNext Enzymatic Methyl-seq Kit (New England Biolabs). Following the deamination step of the workflow, converted bead bound DNA was washed with 80% Ethanol twice on beads.
  • Converted bead bound DNA was amplified with a 6 cycle PCR using primers that added sample identification indices and sequencing adapters for an Illumina machine and a Uracil tolerant polymerase, i.e. Q5U (New England Biolabs) lng of converted DNA was used in allele specific PCR to assess methylation status of the BRCA1 promoter (Swisher et al, 2021). Non-bead bound DNA was used as a control to assess the efficacy of performing the conversion on beads. The yield of the first amplification product was compared to the second amplification product to assess our recovery.
  • Q5U New England Biolabs
  • Table 1 shows the estimated percent recovery of biological DNA following capture on Streptavidin beads, enzymatic conversion of unmethylated Cytosines to Uracils, and amplification by PCR.
  • a medical professional obtains isolated ctDNA from a subject, and uses the methods described in any of previous embodiments to detect the relative abundance of ctDNA and any chemical modifications to the DNA.
  • the medical professional administers a treatment to the subject and monitors the relative abundance of the ctDNA and any chemical modifications, mutations, or any combination thereof to determine the success of a treatment regimen.
  • Boonstra, P. A. et a ⁇ Clinical utility of circulating tumor DNA as a response and follow-up marker in cancer therapy. Cancer and Metastasis Reviews. 2020. 39, 999-1013.

Abstract

Sont divulguées des méthodes de réalisation d'une procédure médicale par détermination de la mutation et de l'état de modification de l'ADN d'un échantillon biologique provenant d'un sujet, par rapport à un échantillon unique. Sont également divulguées des méthodes de diagnostic par la détermination du fait qu'un ADN d'un échantillon biologique contient des modifications et des mutations de nucléobase, par évaluation de la modification de l'ADN et de l'état de mutation par rapport à un échantillon unique.
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